The first report on microbial conversion of steroid compounds was published as early as in 1937 when Mamoli and Vercellone (Ber. 70, 470 and Ber. 70, 2079) described the stereospecific reduction of 17-ketosteroids to 17-.beta.-hydroxysteroids. In 1952, Peterson and Murray (U.S. Pat. No. 2,602,769) described a commercially important process for the 11-.alpha.-hydroxylation of progesterone by Rhizopus nigricans.
Since that time, a great variety of conversions of steroid compounds by means of microorganisms have been described (see, for ex. W. Charney and H. L. Herzog, Microbial Transformation of Steroids, Academic Press, New York, 1967).
Despite this great number of proposals for the microbial transformation of steroid compounds, only a few of these reactions are suitable for the production of important steroid hormones from starting materials which are readily accessible and available in large amounts. In this connection, the sterol compounds contained in 17-C-alkyl side chains of plant or animal origin are to be mentioned primarily, for example, sterols of the cholestane, campestane and stigmastane series.
It has been known that a great variety of microorganism strains, e.g. those of Achromobacter, Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Flavobacterium, Microbacterium, Micybaterium, Nocardia, Protaminobacter, Serratia and Streptomyces are capable of growing on sterol compounds of the kind mentioned, e.g. on cholesterol, as the carbon source (see for ex. Arima et al, 1969, Agr. Biol. Chem. 33: 1636-1643). In doing so, both the ring system and any side chains, which may be present on the ring system are attacked as a rule in a non-specific manner by the microorganisms or degraded in their growth, the attack and degradation in the ring system being predominantly the preferred and/or faster reaction. A recently published paper by Arima et al, Agric. Biol. Chem. 42(2), 411-416 reports on work for the transformation of cholesterol by microbial side chain degradation in the presence of inhibitors to inhibit the ring degradation in the steroid structure.
According to this publication, a considerable proportion of the about 200 wild strains having been tested was capable under the influence of the inhibitors to lead to largely selective side chain degradation in C-17 of the starting material. Some of the strains lead to 3-oxopregna-1,4-diene-20-carboxylic acid (.DELTA.-1,4 BNC) in varying yields.
Japanese Pat. No. 152800 Arima, relates to 3-oxobis norchola-1,4-dienic acid as being useful as a raw material for preparing hormonal steroids. It is prepared by culturing a microorganism capable of converting sterols or their derivatives with dehydrogenation A ring Nocardia cholarina IFO 3338, N. erythropolis NI 9110 FERM-P 3315, Mycobacterium abium IFO 3082, Protaminobacter alboflavus IAM 1040, Bacillus roseus IAM 1257. Typical microorganisms are Mycrobacterium lacticum IAM 1640 FERM-P 3317, Serratia marcescens IAM 1225 FERM-P 3316 etc. The medium contains sterol (e.g. cholesterol, beta-sitosterol, gamma-sitosterol, campesterol, ergosterol, stigmasterol etc.), or an A-ring dehydrogenated derivative (e.g. 4-en-3-one compounds, 1,4-dien-3-one compounds etc.). It also contains a chelating agent capable of forming a complex salt with iron or copper (e.g. 1-nitroso-2-naphtol, salicylaldoxime, chromotoropic acid, alpha,alpha'-dipyridyl, 8-oxyquinoline, nitroso.phenylhydroxylamine, 1-nitroso-2-naphtol-3,6-disulphonic acid, o-phenthroline, tetrahydroxyanthraquinone, ethylenediaminetetraacetic acid, et.) or a nickel or cobalt ion.
The starting sterol or a dehydrogenated derivative is added to the medium in the form of fine powders or solutions in solvents or suspensions in water.
East German Pat. No. 195528 relates to the production of 20-carboxy-1,4-pregnadien-3-one (I) carried out by treating sterols and their 3-substituted derivatives with sterol-degrading microbial cultures under controlled fermentation conditions in the presence of inhibitors for suppressing cleavage of the steroid ring structure.
Due to the potential importance of natural sterol compounds having 17-C side chain or plant original (phytosterols) or animal origin (cholesterol) as starting material for high quality pharmaceutic compositions having steroid basic structure, a great number of attempts have been made to achieve selective side chain degradation on the microbiological route. A summarizing review of these works is found in Adv. Appl. Microbiol. 22, 29 (1977) 29-58, Christoph K. A. Martin "Microbial Cleavage of sterol Side Chains". Reference is made especially to Chapter IV of this publication which classifies the three methods used heretofore for the selective side chain cleavage under A to C. Accordingly, the prior art proposals fall within the following three groups: Transformation of the ring structure of the sterol starting compound in such a manner that the mechanism of ring cleavage is prevented so that the selective side chain cleavage becomes possible; concomitant use of inhibitors to inhibit the steroid ring degradation and or the growth of the microorganism, and, finally the search for microorganism mutants which lead to the desired, as extensive a selective side chain degradation as is possible.
According to said process of prior art, the final products obtained predominantly of the microbial degradation are 17-ketosteroid compound which, while valuable as intermediates for the technical production of hormones of the estrane-androstane- and spirostane-series, are less suitable for the preparation of steroid hormones of the pregnane series, e.g. progesteron, hydrocortison, cortison, prednison, prednisolon, triamcinolon and the like. In the latter cases, it is necessary to incorporate again chemically in 17-position stereospecific side chains.
Heretofore, it was possible only occasionally to obtain as metabolism products of microbial degradation steroid compounds which contain an alkyl side chain group and especially the residue of .alpha.-propionic acid in 17-C position. Steroid compounds of this type would be useful especially for the commercial-scale production of steroid hormones of the pregnane series from sterols of natural origin. It is evident in this connection that the recovery of partial sterol degradation products of this kind is particularly difficult because this requires not only the selective side chain cleavage with simultaneous prevention of breaking-up and degradation of the ring structure, but additionally the growth of the microorganisms with cleavage of the side chains must be ceased in a stage which, according to knowledge gained so far, is merely a transitory station until the 17-ketosteroid structure has been reached. J. M. Whitmarsh describes in The Biochemical Journal, 90, 1964, 23 p to 24 p, the microbiological degradation of cholesterol with a nocardia culture in the presence of inhibitors such as 8-hydroxyquinoline with formation of small amounts of 3-oxo-pregna-4-ene-20-carboxylic acid (.DELTA.4 BNC) and 3-oxo-pregna-1,4-diene-20-carboxylic acid (.DELTA.1,4 BNC). The recently published U.S. Pat. No. 4,029,549 (Antosz et al) describes the microorganism mutant NRRL B-8119 which is said to permit the selective degradation of 17-C steroid compounds having 8 to 10 carbon atoms in the 17-C alkyl group to form 9.alpha.-OH AD and 9.alpha.-OH BN acid. However, as is shown especially by chapter IV C of the publication by C. K. A. Martin, loc.cit., pp. 50 to 52, there exists up to-date no reliable method for the microbiological production of 17-C-steroid-.alpha. -propionic acid compounds from 17-C-side chain steroid substrates or for the reproducible recovery of microorganism defective block mutants which are capable of furnishing 17-C steroid-.alpha.-propionic acid compounds in a reliable manner and in high yields as the end product of enzymatic degradation even if the process is to be operated in the absence of inhibitors of the kind mentioned. However, operation in the absence or in the presence of only small amounts of the inhibitors must appear to be desirable for technical reasons, e.g. because of the desired high space-time yields.
It is an object of the invention to permit the production of 17-C-steroid-.alpha.-propionic acid compounds in commercially useful and improved yields by selective microbial degradation of 17-C-side chain steroid substrata. This is to be achieved with the use of defective block mutant microorganisms which have been prepared and selected in a directive manner and previously recovered from suitable wild strains. In particular, it should be possible but by no means mandatory in this process to use the selected defective block mutants also in the absence of inhibitors which inhibit the steroid ring degradation and/or the growth of microorganisms. Another object of the invention is the safe and reproducible production of the defective block mutant microorganisms which are suitable for the improved, in particular inhibitor-free commercial recovery of 17-C-steroid-.alpha.-propionic acid compounds from sterols of natural origin.
In a narrower sense, a particular object of the invention is the microbial production of 3-oxo-pregna-4-ene-20-carboxylic acid (.DELTA.4 BNC) and/or 3-oxo-pregna-1,4-diene-20-carboxylic acid (.DELTA.1,4 BNC) by the method described herein. The starting material for the microbiological production of these compounds should be in particular natural and/or vegetable sterols which, as waste products, have hardly gained any practical importance up to the present. Simple derivatives of these natural starting materials are also contemplated as suitable starting materials for the invention.
In a particular embodiment, it is an object of the invention to provide methods of producing highly active defective block mutant microorganisms for the particular sterol starting material used in each given commercial-scale process thereby permitting the adaptation of the sterol starting material and the defective block mutant microorganism to be selected. The products of the process according to the invention .DELTA.-4 BNC and .DELTA.1,4 BNC are valuable intermediate products for the production, for ex., of compounds of the progesterone series.